Our long-term objective is to predict how molecular interactions are translated into gene expression in eukaryotic cells. In eukaryotic genomes, transcriptional regulation is strongly affected by nucleosomes which function to compact DNA and to regulate access to it by simple physical occlusion and by providing the substrate for numerous covalent epigenetic tags. We have recently developed a DNA mechanics-based nucleosome model capable of reproducing in vitro free energies of nucleosome formation with high accuracy. We propose to apply this model to predicting nucleosome positions genome-wide, in S.cerevisiae and other model organisms. We will develop descriptions of chromatin structure that incorporate histone octamers competing with other DNA-binding factors for regulatory sequence. Our preliminary results indicate that this competition may be as important for shaping in vivo chromatin structure as intrinsic nucleosome sequence preferences. We will investigate the accuracy of our predictions by exploring the link between nucleosome positions and gene expression in model systems. We propose to construct promoter sequences that incorporate transcription factor binding sites into a computationally designed nucleosome occupancy profile. These constructs will be assayed for levels of gene expression, providing direct insight into the regulatory role played by nucleosomes. In addition, we propose to carry out high-throughput sequencing of nucleosomes reconstituted in vitro on both genomic and chemically synthesized sequences. This data set will allow us to disentangle intrinsic sequence preferences from in vivo effects, and will enable us to improve the purely structure-based DNA mechanics model in a systematic way. Finally, we propose to carry out microarray and large-scale sequencing studies of the dynamic response of chromatin structure to environmental and genetic perturbations. The proposed studies will significantly enhance our understanding of the connection between regulatory DNA sequence, chromatin, and gene expression. PUBLIC HEALTH RELEVANCE: The ability to predict how chromatin structure affects gene expression will open a novel pathway towards numerous applications in biology and medicine, including rational drug design and new, chromatin-based approaches to rewiring cellular networks. The ability to exercise precise transcriptional control over the amount and the type of proteins produced by the cell through making directed changes in chromatin structure will find many uses in bioengineering and synthetic biology, including production of synthetic hormones, enzymes, and therapeutic agents.